A major goal of our laboratory is to understand the molecular details of vitamin A (retinol) processing within the retinoid cycle as well as the consequences of side reactions that occur between retinal and various biological molecules. A variety of serious diseases result from defects in the retinoid (visual) cycle or abnormalities in retinal clearance. Understanding of the fundamental biochemical processes underlying these diseases is essential for the development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of molecular events leading to light perception. Continuity of vision depends on constant regeneration of all-trans-retinal back to its cis isomer in a process known as the retinoid cycle. This series of reactions takes place in photoreceptor and RPE cells. Retinal, the initial substrate and final product of this cycle, is highly chemically reactive and can form toxic conjugates with proteins and lipids. Many visual disorders are caused by malfunctions of the retinoid cycle. Thus, much experimental effort has been devoted to elucidating enzymatic steps comprising the retinoid cycle and all-trans-retinal-mediated retinal degeneration. These studies have delineated key steps of the retinoid cycle. Nevertheless, many important details regarding chemical transformations of retinal and its derivatives are not well understood and many proteins involved in 11-cis-retinal regeneration still await structural, biochemical and functional characterization. This proposal aims to significantly improve our understanding of retinoid cycle transformations at the molecular level as well as to delineate all-trans-retinal-mediated retinal degeneration and the mechanisms that exist in vivo to prevent it. First, we will use a number of biophysical and biochemical techniques to characterize the structure and catalytic mechanism of RPE65, the enzyme responsible for transforming all-trans-retinyl esters to 11-cis-retinol. Second, we will determine the structural and functional properties of LRAT, a crucial enzyme of the retinoid cycle that converts all-trans-retinol to all-trans-retinyl esters. Third, we will purify ABCA4, the photoreceptor-specific ABC transporter that prevents accumulation of the toxic compounds, all- trans-retinal and its derivatives, including N-retinylidene-PE, inside rod outer segment disks. We then will investigate its structure, posttranslational modifications and mechanisms that regulate its activity. Finally, we will identify the mechanisms of all-trans-retinal-induced retinal degeneration by dissecting out those signaling events that lead to the death of retinal cells. PUBLIC HEALTH RELEVANCE: The visual cycle consists of a sequence of enzymatic reactions occurring in photoreceptor and retinal pigment epithelium cells that continuously regenerate the visual chromophore, 11-cis- retinal. Insights derived from analysis of critical protein components of this cycle will significantly improve our understanding of its principal reactions and the consequences of its toxic byproducts that accumulate in disease states.